The long term goal of this research is to understand the molecular basis of protein trafficking between membrane-bound compartments in human cells. Protein transport involves cargo collection into vesicles, vesicle budding, motility, tethering and docking at the target membrane, and subsequent fusion. Tethering and docking are the least understood steps in membrane traffic. The specific goal of this application is to investigate the molecular function of a protein named GCC185, a 185K, trans Golgi network (TGN)-localized protein of the GRIP domain family. We seek to test the hypothesis that GCC185 functions as a tethering protein for transport vesicles arriving at the TGN. With regard to GRIP domain-family Golgins, the most important questions that need to be resolved are: 1. Are these proteins actually tethers? 2. What molecules do these proteins partner with to achieve tethering? 3. Do these proteins act as vesicle-associated tethers or target-associated tethers? 4. Do these proteins bind to TGN-specific SNARE proteins, and how are they released from the Golgi during or after vesicle fusion? To begin to address these questions, we propose to: 1. Use biochemical approaches to determine the molecular basis for GCC185 Golgi complex localization; 2. Test a model for GCC185 as a vesicle-bound tether by accumulating transport vesicles in SNARE- depleted cells; 3. Characterize the binding of GCC185 to SNARE proteins implicated in endosome to Golgi transport, and test whether GCC185 catalyzes SNARE complex formation at the TGN; 4. Establish a membrane tethering assay using purified, immobilized GCC185 to explore its function. This is important because it will establish that GCC185 is a bona fide tethering protein, and may help us to purify these transport carriers for the first time. These experiments will provide important clues to the mechanism by which GCC185 is localized to the trans Golgi network, and what this protein does there, to facilitate the docking and fusion of transport vesicles, inbound from late endosomes. This work has broad implications for our understanding of vesicle docking and fusion events within the secretory and endocytic pathways that are essential for normal human health and disease. [unreadable] [unreadable] [unreadable]